The elucidation of mechanisms of synthesis, processing and transport of viral components at the molecular level will help us to understand mechanisms that govern biosynthesis of normal cellular components and to understand problems of defective viral assembly implicated in some neurological diseases. To study the biosynthesis of viral macromolecular components, we have prepared a battery of monoclonal antibodies reacting with different sites of polypeptides of two negative stranded RNA viruses, Vesicular stomatitis virus (VSV) and measles virus, polyclonal antibodies made against synthetic peptides corresponding in sequence to portions of the viral polypeptides, and genes coding for some of the viral polypeptides of the Vesicular stomatitis virus cloned into convenient expression vectors. One part of the project concerns the study of assembly processes in living rather than fixed or fractionated cells. Purified antibodies are microinjected into cultured cells in order to interfere with specific assembly mechanisms. Microinjection of antibodies labeled with a fluorescent tag is used to track the transport of some viral components, and low light intensity video microscopy in combination with differential interference contrast microscopy allows us to document on videotapes the transport of labeled antibodies marking the transport of viral components. Immunocytochemistry at the electron microscopic level is used to determine the ultrastructural localization of injected antibodies. A second part of the project concerns the elucidation of mechanisms which leads to the ultimate assembly of viral components at the sites of viral budding. High resolution stereo views are obtained from platinum-carbon replicas of the outer and the inner side of the plasma membrane of cells infected with either one of the above mentioned viruses. The location of specific viral components at the plasma membrane are marked with antibodies coupled to an electron dense marker, colloidal gold. Temperature-sensitive viral mutants with lesions in polypeptides affecting normal budding are studied to obtain systems in which viral budding can be synchronized.